CN117653653A - Small molecule nucleic acid medicine for intervening cancer specific LINC01419 and application thereof in liver cancer targeted therapy - Google Patents
Small molecule nucleic acid medicine for intervening cancer specific LINC01419 and application thereof in liver cancer targeted therapy Download PDFInfo
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Abstract
The invention provides a LINC01419 targeted siRNA, which comprises 2'-OMe, FC (fluoro), FU (fluoro) chemical modification, so that the in vivo stability is improved, and the 3' -end of the siRNA is coupled with GalNAc, so that the liver targeting of the siRNA is enhanced. The invention also provides application of the reagent for detecting the LINC01419 level in preparing a liver cancer prognosis evaluation kit.
Description
Technical Field
The invention relates to the field of tumor treatment, in particular to a small molecule nucleic acid drug for intervening cancer specific LINC01419 and application thereof in liver cancer targeted therapy.
Background
Long non-coding RNA (lncRNA) is a type of non-coding RNA with a sequence of more than 200 nucleotides, which has no or very weak protein-coding ability and which functions biologically in cells mainly in the form of RNA [1] . As the research on the lncRNA is continued in recent years, the lncRNA is found to be variousThe molecular biological mechanism plays an important role in the biological characteristics of cell proliferation maintenance, cell growth inhibition escape, cell immortalization acquisition, angiogenesis, invasion and metastasis, energy metabolism reprogramming, immune supervision escape and the like in the malignant transformation process of cells [2,3] . Compared with protein coding genes, the lncRNA has more tissue specificity, and the abnormal expression in tumor and the important biological functions and significance thereof greatly expand the potential diagnosis and treatment targets of tumor [4] 。
Intervention of tumor-associated lncRNA expression in cells is also a potential tumor treatment based on abnormal expression of lncRNA in tumors and diverse molecular mechanisms that exert a pro-cancerous function. Related pathways include: degrading the lncRNA transcript by specific siRNA, ASO, aptamer and ribozyme; modulating transcription of the lncRNA by altering its promoter activity by inhibiting transcription factors that bind to the lncRNA gene promoter; developing small molecules or polypeptides that block the binding of lncRNA to other proteins, DNA, RNA, or complexes by binding to specific binding pockets; designing nucleic acid aptamer, disrupting lncRNA function by targeting lncRNA at specific structural regions, blocking its binding to interacting molecules, and the like [5,6] . RNA therapies using mainly antisense oligonucleotides (ASOs) and small interfering RNAs (sirnas) have evolved well in recent years, and several drugs have been FDA approved, which are of great significance for the treatment of tumors and a variety of other diseases. siRNA-induced RNA interference technology has great potential in the treatment of tumors, infectious diseases, nervous system diseases and the like [7] . However, siRNA as a drug to exert an interfering effect in vivo requires realization of endocytosis, lysosomal escape, and avoidance of degradation by nucleases, requiring high stability; there is also a need to overcome the barriers in terms of treatment specificity, tolerability and delivery during clinical transformation [8] . N-acetylgalactosamine (GalNAc) is a high affinity ligand of hepatocyte-specific asialoglycoprotein receptor (ASGPR) and can participate in clathrin-mediated endocytosis, so that siRNA enters hepatocytes and has great advantages in the field of liver targeted delivery [9] 。
Patent CN 111485020A discloses the treatment of hepatocellular carcinoma using shRNA interfering with LINC01419, which can be used for general cellular level functional studies by interfering with LINC01419 expression. However, this method further has an effect on normal cells (for example, testis tissue expressing LINC 01419) such as AAV gene therapy in vivo, and has high in vivo toxicity and side effects. Relatively, siRNA treatment is convenient and fast to operate, can achieve a higher efficient gene silencing effect, has small toxic and side effects on cells and tissues, and has a mature delivery mode at present.
Reference is made to:
[1]BATISTA PEDRO J,CHANG HOWARD Y.Long Noncoding RNAs:Cellular Address Codes in Development and Disease[J].Cell,2013,152(6):1298-307.
[2]HUARTE M.The emerging role of lncRNAs in cancer[J].Nat Med,2015,21(11):1253-61.
[3]SCHMITT A M,CHANG H Y.Long Noncoding RNAs in Cancer Pathways[J].Cancer Cell,2016,29(4):452-63.
[4]BHAN A,SOLEIMANI M,MANDAL S S.Long Noncoding RNA and Cancer:A New Paradigm[J].Cancer Res,2017,77(15):3965-81.
[5]SCHNEIDER-POETSCH T,YOSHIDA M.Along the Central Dogma-Controlling Gene Expression with Small Molecules[J].Annu Rev Biochem,2018,87:391-420.
[6]BACH D H,LEE S K.Long noncoding RNAs in cancer cells[J].Cancer Lett,2018,419:152-66.
[7]SASSO J M,AMBROSE B J B,TENCHOV R,et al.The Progress and Promise of RNA Medicine horizontal line An Arsenal of Targeted Treatments[J].J Med Chem,2022,65(10):6975-7015.
[8]WINKLE M,EL-DALY S M,FABBRI M,et al.Noncoding RNA therapeutics-challenges and potential solutions[J].Nat Rev Drug Discov,2021,20(8):629-51.
[9]DEBACKER A J,VOUTILA J,CATLEY M,et al.Delivery of Oligonucleotides to the Liver with GalNAc:From Research to Registered Therapeutic Drug[J].Mol Ther,2020,28(8):1759-71.
disclosure of Invention
To solve the above-mentioned shortcomings, the present invention uses LINC01419 as a research target and GalNAc-coupled siRNA as a drug delivery strategy.
Specifically, the first aspect of the invention provides application of LINC 01419-targeted siRNA in preparing liver cancer drugs.
In certain embodiments, the targeting sequence of the siRNA comprises a nucleic acid as set forth in SEQ ID NO. 6 (CCUCAAUUUCCAUGGCAAUAU).
In certain embodiments, the siRNA synthesis sequence comprises a nucleic acid as set forth in SEQ ID NO. 15 (CCUCAAUUUCCAUGGCAAUAUUU), wherein the 3' end is cantilevered with UU to enhance silencing.
In certain embodiments, the siRNA comprises a 2' -OMe modification (i 2 OMe) and a fluoro modification (i 2F); preferably, the specific modified sequences are characterized by: i2OMeC// i2OMeC// i2OMeU// i2OMeA// i2OMeA// i2FU// i2OMeU// i2FU// i2FC// i2FC// i2FC// i2 OMeA' i2OMeU// i2OMeG// i2OMeG// i2OMeC// i2OMeA// i2OMeA// i2OMeU// i2OMeA// i2OMeU// i2OMeU// i2OMeU/. In certain embodiments, the 3' end of the siRNA is coupled to trivalent GalNAc.
In a second aspect, the invention provides LINC 01419-targeting siRNA whose targeting sequence comprises the nucleic acid set forth in SEQ ID NO. 6 (CCUCAAUUUCCAUGGCAAUAU).
In certain embodiments, the synthetic sequence of the siRNA synthesis sequence siRNA comprises a nucleic acid as set forth in SEQ ID NO. 15 (CCUCAAUUUCCAUGGCAAUAUUU), wherein the 3' end cantilevers UU to enhance silencing.
In certain embodiments, the siRNA comprises a 2' -OMe modification (i 2 OMe) and a fluoro modification (i 2F); preferably, the specific modified sequences are characterized by: i2OMeC// i2OMeC// i2OMeU// i2OMeA// i2OMeA// i2FU// i2OMeU// i2FU// i2FC// i2FC// i2FC// i2 OMeA' i2OMeU// i2OMeG// i2OMeG// i2OMeC// i2OMeA// i2OMeA// i2OMeU// i2OMeA// i2OMeU// i2OMeU// i2OMeU/.
In certain embodiments, the 3' end of the siRNA is coupled to trivalent GalNAc.
In a third aspect, the invention provides the use of a reagent for detecting LINC01419 level in the preparation of a liver cancer prognosis evaluation kit.
In certain embodiments, the reagents comprise primers and/or probes for LINC01419 detection.
In certain embodiments, the primers detected in LINC01419 are set forth in SEQ ID NOs 3 and 4
Compared with the prior art, the invention has the beneficial effects that:
1) The surface of the liver cell membrane can specifically express an ASGPR receptor, and the GalNAc coupled siLINC01419 can specifically bind with the ASGPR receptor on the surface of the liver cell membrane so as to realize targeted delivery of the GalNAc-siLINC01419 into the liver cells through receptor-mediated endocytosis, thereby reducing off-target effect. Moreover, ASGPR is a receptor mainly expressed on the surface of hepatic cell membrane, and about 50 ten thousand ASGPR receptors are present on each hepatic cell membrane surface, and the high abundance of ASGPR in expression can significantly increase the efficiency and effectiveness of targeted delivery.
2) GalNAc coupled siRNA can realize good drug distribution effect through subcutaneous injection, target liver with high efficiency, and compared with intravenous injection, the dosage required is smaller, and the side effect caused by administration is obviously reduced.
3) The siRNA is subjected to chemical modification such as dimethoxy modification, fluoro modification and the like, so that the nuclease activity resistance of the siRNA is enhanced, and the stability of the siRNA is improved.
4) In vivo research results show that GalNAc-siLINC01419 can effectively inhibit in vivo growth and metastasis of nude mouse liver in-situ xenograft tumors. Has high clinical conversion value and market prospect.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 shows expression in different tissues of LINC 01419. The expression level of LINC01419 in human normal tissues was analyzed by GTEx database, and LINC01419 was found to be hardly expressed in human normal tissues except testis (fig. 1A). The expression levels of LINC01419 in 33 tumor tissues were analyzed by TCGA database, LINC01419 was expressed in a variety of tumors, and expression in liver cancer was particularly high (fig. 1B).
FIG. 2 shows that LINC01419 is specifically highly expressed in liver cancer tissue and correlated with patient prognosis. (Cohort 1: TCGA-LIHC dataset, cohort 2:Gepliver dataset,Cohort 3:GSE77314dataset,Cohort 4:GSE144269dataset) the data set data all showed that LINC01419 was significantly highly expressed in liver cancer but not substantially in paracancerous tissue or normal liver (FIGS. 2A-D). Furthermore, LINC01419 high expression was associated with a poor prognosis for patients (fig. 2E).
Fig. 3 shows that LINC01419 was interfered with by multiple sirnas in liver cancer cell line Huh7, respectively, and two optimal qPCR detection interference efficiencies were selected for subsequent experiments (fig. 3a, b). And stable knocking down of LINC01419 by shRNA in Huh7 and PLC cells with high expression of LINC01419, and stable overexpression of LINC01419 in SK-Hep1 and HepG2 cells with low expression of LINC01419, both have good expression efficiency (FIGS. 3C, D).
Figure 4 shows in vitro cell experiments demonstrating that interfering LINC01419 expression by siRNA, and CCK8 proliferation, clonogenic, cell migration and invasion experiments, the ability to significantly inhibit liver cancer cell proliferation, migration and invasion was found to interfere with LINC01419 expression (figures 4A-D).
Figure 5 shows in vitro cell experiments demonstrating that LINC01419 promotes liver cancer cell proliferation, migration and invasion. Cell function experiments were performed by constructing a LINC01419-shRNA stable knockdown cell line and a LINC01419 stable overexpressing cell line. CCK8 proliferation, clonogenic, cell migration and invasion experiments were performed after stable knockdown of LINC01419 in LINC 01419-expressing Huh7 and PLC cells, and it was found that interfering with LINC01419 significantly inhibited the proliferation, clonogenic, migratory and invasion capacity of hepatoma cells (FIGS. 5A-D). In contrast, overexpression of LINC01419 in SK-Hep1 and HepG2 cells with low expression of LINC01419 significantly promoted proliferation, clonogenic, migratory and invasive of hepatoma cells (fig. 5E-H).
Figure 6 shows in vivo cell experiments demonstrating that LINC01419 promotes liver cancer cell proliferation, migration and invasion. In vivo experiments were performed on stably-interfered LINC01419 cell lines using a nude mouse subcutaneous neoplasia model, and the growth rate and tumor weight of both independent LINC01419 knockdown groups of subcutaneous neoplasias were significantly lower than that of the control group (fig. 6a, b). Ki67 staining of tumor tissues also showed a significant decrease in Ki67 positive rate in the LINC01419 knockdown group of tumor cells (FIG. 6C). Meanwhile, huh7 cells stably expressing EGFP-luciferases are constructed, in-vivo transfer experiments are carried out by interfering LINC01419 on the basis, and in-vivo imaging results show that the interfering LINC01419 remarkably inhibits the growth of liver cancer cells in the liver in-situ of nude mice (figure 6D), HE staining and analysis of liver and lung tissues also show that the interfering LINC01419 remarkably inhibits the liver transfer of tumors and the lung transfer is reduced (figure 6E).
FIG. 7 shows a flow chart for establishing a liver in situ xenograft model for GalNAc-siRNA treatment.
FIG. 8 shows the therapeutic effect of GalNAc-silnc 01419 in vivo. The in vivo growth capacity of tumors was significantly reduced in GalNAc-silnc 01419 treated mice compared to control group, and fluorescence intensity was significantly reduced (fig. 8A-B); meanwhile, the degree of staining of Ki67 in tumor tissues of the GalNAc-silnc 01419 treated group mice was significantly reduced (FIG. 8C); HE staining found that GalNAc-silnc 01419 treated mice had significantly reduced intrahepatic and pulmonary transfer capacity compared to control mice (fig. 8D-E).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
Example 1 Experimental methods
1.1RNA sequencing dataset download and analysis
Downloading human normal tissue sample expression data of 29 different tissue types from a GTEx database; downloading from the TCGA database a bam file of RNA-seq of 10358 human tumor samples of 33 different cancer types; TPM expression values of LINC01419 transcripts were quantified by the bioinformatics software StringTie analysis.
For liver cancer tissue expression data, queue 1: tissue expression data from 369 patients (including 50 pairs of paired liver cancer and paracancerous tissues) in the TCGA liver cancer patient RNA-seq dataset (TCGA-LIHC) of GDC data (https:// portal.gdc.cancer/.gov). Queue 2: tissue expression data for 105 HCC patients (including 50 pairs of paired liver cancer and paracancerous tissues) were obtained from GepLiver (http:// www.gepliver.org /). Queue 3: 50 pairs of HCC tissue and paracancestor tissue expression data in the GSE77314dataset downloaded from the Gene expression Integrated database (GEO, https:// www.ncbi.nlm.nih.gov/GEO /). Queue 4: 70 pairs of HCC tissue and paracancerous tissue expression data in GSE144269dataset downloaded from GEO.
1.2 cell culture
Human hepatoma cells Huh7, PLC, SK-Hep1 and HepG2 and human embryonic kidney cells HEK-293T cells were cultured in high-sugar DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% diabody (penicillin and streptomycin). Cell culture at 37℃with 5% CO 2 In an incubator.
Generally, cells can be passaged when they proliferate to a density of 70-80%. During cell passage, the cells to be treated are taken out from an incubator, the culture medium is poured out and added with PBS to clean the cells twice, then a proper amount of pancreatin (0.25%) is added to infiltrate the cells and stand for a moment, the cells are observed to fall off the culture dish by microscopic examination, 1mL of complete culture medium is added to discard pancreatin to lightly blow, the cells are fully suspended, a proper amount of cells are taken and added into a new culture dish, a proper amount of complete culture medium is supplemented to shake uniformly, and the cells are placed at 37 ℃ and 5% CO 2 Culturing in an incubator.
1.3 real-time fluorescent quantitative PCR (qPCR)
Cellular RNA was extracted with TRIzol reagent (Invitrogen, CA, USA). RNA was reverse transcribed into cRNA using Evo M-MLV RT Master Mix (Accurate Biology, hunan, china).
qPCR reactions used SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, huntan, china), 3 multiplex wells per set of samples.
The reaction system (8 μl) was configured in 384 well plates:
after sample addition, 384 plates are sealed, are put into a Quantum studio 5Real-Time PCR System instrument for qPCR reaction after instantaneous centrifugation, and the reaction program is set:
pre-denaturation at 95 ℃ for 15s; denaturation at 95℃for 5s, annealing at 60℃for 30s, and reaction cycle 40.
And after the reaction is finished, deriving a CT value, and taking beta-actin as an internal reference, wherein the calculation mode is delta CT=the CT value of an experimental group (target gene) -the CT value of a control group (internal reference gene), and 2-delta CT is the relative expression quantity of the genes.
TABLE 1 qPCR primer sequences
1.4 siRNA transfection
siRNA is biosynthesized from libo. The siRNA dry powder is added with DEPC water for dissolution after instantaneous centrifugation, prepared into storage solution of 20 mu M, and stored in a low-temperature refrigerator of-80 ℃ or-20 ℃ after split charging.
Cells to be transfected are inoculated into a six-hole plate one day in advance, cultured overnight, and transfected preferably when the density is 50-60% in the next day; incubation of transfection system: mixing 5 μL siRNA with 200 μL opti-MEM, adding 5 μL RNAiMAX into 200 μL opti-MEM, gently stirring, mixing, and standing for 5min; mixing the two systems, gently blowing and beating uniformly, and standing for 15min; then taking out six-hole plate cells to be transfected from the incubator, discarding the culture solution, adding 1.6mL of serum-free and double-antibody-free culture medium, transferring the well-incubated transfection system into each hole, marking, slightly mixing uniformly, and then placing into the incubator for culturing; after 6-8 hours, the culture medium is replaced by 2mL of fresh complete culture medium. Subsequent experiments can be carried out 24-48 hours after transfection.
TABLE 2 siRNA sequences
1.5 lentiviral vector construction
Construction of shRNA plasmid for stably knocking down LINC01419
The shRNA forward and reverse primers were synthesized at gold only Biotechnology Co.
Annealing the primer segment into shRNA double chains; the Lenti-gRNA-Puro (Addgene # 84752) vector was digested with BsmB I-v2 (New England Biolabs, MA, USA); the shRNA was ligated into the Lenti-gRNA-Puro vector by Solution I ligase (Takara, japan) ligation. Then, E.coli HB101 is transformed, after plating, monoclonal expansion culture is selected, plasmid is extracted, and after sequencing and identification are correct, the plasmid construction is completed for subsequent lentivirus packaging.
TABLE 3 shRNA primer sequences
Plasmid construction for stable overexpression of LINC01419
Designing a specific amplification primer according to the full-length sequence of LINC014191, adding 15-20 nt sequences which are overlapped with the two ends of the overexpression vector after enzyme tangentially-cutting at the 5' end of the primer, and amplifying the full-length fragment of LINC01419 by using PrimeSTAR HS Premix high-fidelity enzyme (Takara, japan) PCR; the pCDH-CMV (Addgene # 72265) vector was digested with EcoR I-HF (New England Biolabs, MA, USA) and BamH I-HF (New England Biolabs, MA, USA); the full length fragment of LINC01419 was ligated into pCDH-CMV vector by ClonExpress Ultra One Step Cloning Kit (Vazyme, nanjin, china) seamless cloning; and after the transformation plasmid is sent to sequencing and identification, the LINC01419 over-expression plasmid is successfully constructed, and the empty Vector without the LINC01419 fragment is used as a Vector negative control.
LINC01419 full-length sequence (SEQ ID NO: 14):
AACCCGCTCGGGTCCCCTTCCACATCGTGGAAGCTTTGTTCTTTCGCTCTTTGCAATAAATCTTGCTACTGCTCACTCTTTGGGTCCACGCTGCTTTTATGAGCTGTAACACTCACCGCGAAGATCTGCAGCTTCACTCCCGAGCCAGCGAGACCACGAACCCACCAGAAGGAAGAAACTCCGAACACATCTGAACATCAGAAGGGCAGACTCCAGACACGCCACCTTAAGAGCTGTAACACTCACCGCGAGGGTCCACGGCTTCATTCTTGAAGTCAGTGAGACCAAGAACCCACCAATTCCGGACACAATTTCTTGGCTCTCAGTGGCTTCCATGGAAGTTCTGCAATCAACCAGCAGGAGAACCGCTTAAACCCAGGAGGCGGAGGTTGCAGTGAGCCAAGTATGCATCACTGCACTCCAGCCTGGAAGACAGAGTGAGACCCTGTCTCAACAAAATAAATTAAAATAAAAAATAATATATTTTTCTAACTATCATCCCTTTTCCAAATCAGGAATTCCCCTTAAGTTTTCCTCAATTTCCATGGCAATATCTTTGCATAGATTCATTAAGAATTTGTCCTTTTTAAATAAAAAATATAAAGGGAACTATTCATTAGGCAACAAATGCCTTGTCTGAAATATCACATTTGAGAATGCTGCTCATTTAATCAGAAAGGTACGCTACTTTAAAGAACTGAGGTCCACTTTCTGGAGCCAAAAACTCATAAATCCCTCTCAGAAAAACCTGATTTGCTTTGTAGGGTCTCAGGTTTAGAGATGCTGAAAAAGATATTTTCGTTGCAGACAAAGGACCTCAGAGTATTTGGAGAACTTTGAGAAGAGAGGAATTCTCCCAAATGTATAGGTGTCACAGGTAAAATACAGTCGAGAGATTTTCTTGGACTTTAATTCCTTAAATCAGGATAGCAAATAATAGGGGCTTTTACAAATTCAATCTGTTTCCTTACAAAAATTTTCAGCAAAGTAATTTCAGCAAATTAATTTTCAGCAAAGTAAATGTAAGAAGACTTATGTGAAAAATTAACATTCTCCATGTATCTATGAAGCCAAACCTAATAAAACCAGCTTTAATTTGTGCTCAAGAATATTATTTCACTGAGTTTTCTTAAATCACAAAGGGGAGACTGTTATGAAAACTGATATAAAATAAAAAAAACAAGGAAGAAAAGTCTACTAGATGTCTCTAATGGAAGACTGCATTTTTAGAACATATCCTTATAGGCGATTCTAGCCTTTCTCTGCTATTTGGCTCTCACACTCTTTACCGTGCAGATAATTCACAGCAATGCAAAAGAATCCTCATCTATAGCCATGAAAATAAGTTATTTGTTATTTCTGGTAAAGGTTCAATTGACCTCCCCTTCCAGGATGAAGAAAGTTTCATGTCTTTCTGCATCATTTCAACTATTCCTTACTACATATAAATCTGCACTTGTTAACTTCTATTTTGAATTGATTGTGGCATCTGCCTGCTTCCCCATTAAAACTGAATAAAATCTTTAACACATAAAAA。
pWPXL-EGFP-Luciferase plasmid construction for mouse in vivo imaging experiments
Cloning a Luciferase gene (Luciferase) into a pWPXL vector (Addgene # 12257), wherein the pWPXL vector itself contains EGFP, and the Luciferase is inserted behind the EGFP and is subjected to fusion expression, so that a pWPXL-EGFP-Luciferase plasmid is obtained.
1.6 lentivirus packaging and infection, construction of stably expressed cell lines
Lentivirus packaging: after the HEK-293T cells with good growth state are digested by pancreatin, a proper amount of cells are inoculated into a 6-hole plate, and after the cells are cultured overnight, the density reaches 70-80%, slow virus packaging can be carried out; 2 mu g of target lentiviral vector plasmid, 1.4 mu g of packaging plasmid psPAX2 (Addgene # 12260), 0.7 mu g of envelope plasmid pMD2.G (Addgene # 12259) and 400 mu L of serum-free DMEM culture solution are mixed, another EP tube is used for mixing 10 mu L of liposome transfection reagent and 400 mu L of serum-free DMEM culture solution, and the mixture is left standing for 5min at room temperature; mixing the two systems, lightly blowing and mixing, and standing for 15min; taking out 6-hole plate HEK-293T cells from the incubator, discarding the culture solution, adding 1.2mL of serum-free culture medium into each hole, slowly adding the well-incubated transfection system along the hole wall, uniformly mixing, and returning to the incubator; after culturing for 6-8 h, changing the culture solution into 2mL of fresh complete culture medium; collecting culture medium after 48h transfection, sucking out the culture medium by a syringe, filtering with a 0.45 μm filter membrane, and collecting in an EP tube to obtain virus liquid which can be directly used for infecting cells or stored in a refrigerator at-80 ℃.
Lentiviral infection and stable strain construction: when the cell density in good condition reaches 50-70%, slow virus infection can be carried out, taking six pore plates as an example, replacing fresh culture solution by cells, adding polybrene with the final concentration of 6 mug/mL, and putting back into an incubator; after 30min, taking out the cells, adding a proper amount of virus liquid into the holes, and replacing fresh culture medium after overnight infection; after 36-48 h, observing the infection efficiency of the fluorescent plasmid by using a fluorescent microscope and then carrying out flow sorting; the resistance screening plasmid can be added with a corresponding screening drug, RNA detection efficiency can be extracted after screening, and a cell function experiment can be directly carried out by confirming a stable expression strain with better efficiency, and the stable cell strain is frozen at the same time.
1.7 in vitro cell function experiments
1) CCK8 cell proliferation assay
Cells in good condition were digested with pancreatin, the cells were homogenized with serum-free medium, transferred to EP tubes, 10 μl of cell suspension was aspirated after gentle homogenization, mixed with 10 μl of trypan blue (Invitrogen, CA, USA), 10 μl was added to Countess cell counting chamber slides (Invitrogen, CA, USA) after homogenization, and counted in an automatic cell counter. Diluting cells to 1X 10 with complete medium based on cell count 4 After being blown uniformly, the mixture is inoculated into 96-well cell culture plates with 100 mu L of each well by a row gun and placed into an incubator for culture. After the cells adhere to the wall, CCK-8 cell proliferation activity detection is carried out on days 1, 3 and 5 respectively. The culture medium of the well to be measured is discarded, 100. Mu.L of the mixture containing 10% ofComplete medium of CCK8 reagent (MCE, shanghai, china). And (5) placing the strain in an incubator for further culture for 2 hours, and then placing the strain in an enzyme-labeled instrument for detection of OD450nm.
2) Cell clone formation assay
Cells in good condition were counted after digestion and the cell suspension concentration was adjusted with complete medium. 100. Mu.L of each well (1000/2000/3000 cells) was then added to each six-well plate. 3 multiple wells of each cell, adding 2mL of complete culture medium into each well, mixing uniformly, and placing into an incubator for culturing. An appropriate amount of culture medium may be added after a few days apart to supplement consumption. Culturing for 8-14 days, observing the clone formation condition under a lens, discarding the culture solution if the clone grows well, washing twice with PBS, sucking the residual PBS liquid, adding 0.1mg/mL crystal violet solution prepared by methanol, standing and dyeing at room temperature for 15min, sucking the crystal violet solution, fully washing with tap water, airing at room temperature, and scanning for imaging. The number of clones was counted based on the scanned image, and the difference between the experimental group and the control group was analyzed.
3) Cell migration and invasion experiments
Cells in good condition were digested with pancreatin, resuspended in serum-free culture medium, transferred to EP tubes, and cell concentration was adjusted with serum-free culture medium after cell counting. Huh7, PLC and SK-Hep1 cells were adjusted to 2.5X10 5 Per mL, hepG2 cells were adjusted to 5X 10 5 /mL. mu.L of complete medium was added to the 24-well plate in advance, and then a transwell chamber (Falcon, new Jersey, USA) was placed, and 200. Mu.L of diluted cell suspension was added to the chamber. The 24-well plate was placed in an incubator for culturing, huh7 and PLC cells were cultured for 14h, sk-Hep1 cells were cultured for 12h, and hepg2 cells were cultured for 48h. And after the culture time is over, taking out the cell, putting the cell into crystal violet dye solution prepared by methanol, dyeing for 15min, washing with clear water, wiping the inner side of the cell with a cotton swab, and airing after full washing. The dried cells were photographed and collected with an inverted microscope, and each cell was photographed with a random selection of 3-5 fields of view, and then counted with Image pro plus software.
For cell invasion experiments: matrigel (BD, new Jersey, USA) was thawed in a refrigerator at 4 ℃ and on ice at 1: ratio 9 diluted Matrigel was prepared with serum-free medium. Then 600. Mu.L of complete medium was added to the 24-well plate, a transwell chamber was placed, 60. Mu.L of diluted Matrigel was added to the chamber, spread evenly with a gun head, and then the 24-well plate was placed in an incubator for 1-2 hours, followed by subsequent experiments. Cell invasion requires doubling the concentration of the cell suspension compared to that required for cell migration experiments, or prolonged culture time, other steps are the same as for cell migration experiments.
1.8 subcutaneous Oncomelania test in nude mice
Female BALB/c nude mice of 5 weeks of age were kept in an SPF environment and supplied with sufficient food and water during growth. Experiments were performed on three groups of 6 mice each, each injected with 3X 10 cells 6 (200. Mu.L in volume). After the cells are extracted by using a 1mL syringe, the redundant air in the syringe is exhausted, the cells are injected under the right armpit of the mouse (the epidermis is sterilized by alcohol cotton balls before injection), the tumor volume and the weight of the mouse are measured every 3 days after 7-10 days of tumor formation, and the calculation formula of the volume is 0.5 xL xW 2 (L is length and W is width). 3-4 weeks until the tumor volume is less than 2000mm 3 When the experiment is finished, the subcutaneous tumors are peeled out after the mice are euthanized, weighed and recorded, and all the subcutaneous tumors are placed in groups for photographing recording. And (3) washing the fresh tumor tissue in PBS, cutting a small piece, adding a TRIzol reagent, and extracting RNA of the subsequent tissue. The excess tissue was fixed with 4% paraformaldehyde for subsequent paraffin embedding, paraffin sectioning and immunohistochemical staining.
1.9 in situ liver transplantation experiment in nude mice
Female BALB/c nude mice of 5 weeks of age were three groups of 6, each injected with 1.5X10 6 Huh7 cells stably expressing EGFP-Luciferase (50. Mu.L in volume, matrigel mixed with empty medium 1:1). The cell suspension was injected into the mouse liver by in situ transplantation of nude mouse liver. During this period, observation of the mice' status ensured that the tumor burden was not excessive, and after 4 weeks, each mouse was anesthetized by inhalation of isoflurane (RWD, shenzhen, china) after intraperitoneal injection of 150mg/kg D-Luciferin (Yeasen, shanghai, china) and then imaged with a IVIS Lumina LT Series III in vivo imaging system to collect pictures. After the imaging is finished, the mice are euthanized, and the livers of the dissected mice are photographed and stored after being isolatedSmall pieces of tumor tissue were left for RNA extraction, and then mouse liver and mouse lung were fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin (H&E) Dyeing.
1.10 GalNAc-silnc 01419 treatment of nude mouse liver in situ transplantation tumor model
GalNAc-siRNA lake Hema biosynthesis for in vivo animal experiments.
GalNAc-siNC targeting sequence UUCUCCGACGUGUCACGUUU (SEQ ID NO: 16) was biosynthesized by hippopotamus. GalNAc-silNC 01419 targeting sequence was CCUCAAUUUCCAUGGCAAUAU (SEQ ID NO: 6). (GalNAc-silnc 01419 targeting sequence the silnc 01419-1 corresponding sequence was selected according to the most obvious set of siRNA interference efficiency and function in vitro cell experiments).
GalNAc-siNC and GalNAc-siLINC01419 were chemically modified with 2' -OMe, FC (fluoro), FU (fluoro). The sequence after modification of the GalNAc-SiNC sense strand is: i2OMeU// I2OMeC// I2FG// I2OMeA// I2FA// I2FC// I2FG// I2FA// I i2OMeU// i2OMeG// i2OMeU// i2OMeC// i2OMeA// i2OMeC// i2OMeG// i2OMeU// i2OMeU/. The modified sequence of GalNAc-siLINC01419 is: i2OMeC// i2OMeC// i2OMeU// i2OMeA// i2OMeA// i2FU// i2OMeU// i2FU// i2FC// i2FC// i2FC// i2 OMeA' i2OMeU// i2OMeG// i2OMeG// i2OMeC// i2OMeA// i2OMeA// i2OMeU// i2OMeA// i2OMeU// i2OMeU// i2OMeU/. And the 3' -end of both siRNA sense strands is coupled with trivalent GalNAc molecules. Wherein siRNA can enhance the silencing effect of siRNA by adding 3' cantilever UU (Elbashir SM et al, EMBO J.2001Dec 3;20 (23): 6877-88.Doi:10.1093/Emboj/20.23.6877.PMID: 11726523).
Construction of a cell line stably expressing EGFP-Luciferase: and (3) infecting Huh7 cells by using pWPXL-EGFP-Luciferase virus, collecting cells after 48 hours, performing cell flow sorting to obtain GFP positive cells, and further performing expansion culture to obtain liver cancer cell lines stably expressing EGFP-Luciferase.
12 male BALB/c nude mice of 6 weeks of age were injected 5X 10 each 6 Huh7 cells stably expressing EGFP-Luciferase (50. Mu.L in volume, matrigel mixed with empty medium 1:1). The cell suspension was injected into the mouse liver by in situ transplantation of nude mouse liver. After 7 days of in situ implantation, each mouse was intraperitoneally injected with 150mg/kg D-Luciferin (Yesen, shanghai, china) was then inhaled for isoflurane (RWD, shenzhen, china) anesthesia and then imaged with a IVIS Lumina LT Series III in vivo imaging system to acquire images. Nude mice were randomized into two groups (n=6/group), and treatment experiments were performed subcutaneously with GalNAc-siNC and GalNAc-silnc 01419 (5 mg/kg), once a week, for two consecutive weeks, and weekly with in vivo imaging. After the third week of imaging, euthanizing the mice, dissecting the livers of the mice, taking pictures for storage after the in vitro, taking small tumor tissues for RNA and protein extraction, and fixing the livers of the mice and the lungs of the mice in 4% paraformaldehyde for subsequent H&E staining and immunohistochemical staining.
EXAMPLE 2 experimental results
2.1 expression of LINC01419 in liver cancer
The expression level of LINC01419 in human normal tissues (the sample amounts of each tissue type are noted after the sample names in the figures) was analyzed by GTEx database information, and LINC01419 was found to be hardly expressed in human normal tissues except testis (fig. 1A). The expression levels of LINC01419 in 33 tumor tissues (the tissue sample amounts of each tumor type are labeled after the tumor names in the figures) were analyzed by TCGA database, LINC01419 was expressed in various tumors, and expression in liver cancer was particularly high (fig. 1B). The above results indicate that LINC01419 is a cancer-specific expressed molecule.
2.2LINC01419 expression in liver cancer tissue and prognostic relevance
The RNA sequencing data of the multiple data sets of coort 1:tcga-LIHC dataset, coort 2:Gepliver dataset,Cohort 3:GSE77314dataset,Cohort 4:GSE144269dataset (RNA sequencing data expression profile data of the samples were fixed, coort 1:n=50, coort 2:n=50, coort 3:n=70, coort 4:n=70) all showed significantly high expression of LINC01419 in liver cancer but substantially no expression in paracanced tissues or normal liver (fig. 2A-D), providing a good target for specifically targeting liver cancer cell therapy. And LINC01419 high expression correlated with a worse prognosis of the patient (fig. 2E), further improving the significance of targeted LINC01419 treatment.
2.3 in vitro experiments to study the correlation of LINC01419 with liver cancer cell proliferation, migration and invasion
To investigate the biological function of LINC01419, four sirnas specifically targeting LINC01419 were designed (see methods for sequences) and transiently transfected into Huh7 cells expressing LINC01419, respectively, and after 48h, the extracted RNAs were tested for interference efficiency of LINC01419 by qPCR (fig. 3A). Two siRNAs with highest efficiency are further selected for subsequent functional study. Both sirnas were transiently transfected in high expressing Huh7 and PLC cells with good interference effects (fig. 3B). CCK8 proliferation, clonogenic, cell migration and invasion experiments were then performed in cells of siRNA-interfered LINC01419, and it was found that interfering LINC01419 significantly inhibited the proliferation, clonogenic, migration and invasion capacity of hepatoma cells (fig. 4A-D). The functions of the cells are explored by constructing cell lines which stably interfere and overexpress LINC01419, the constructed LINC01419-shRNA plasmid and LINC01419 over-expression plasmid are subjected to slow virus packaging, the cells are infected with virus liquid overnight, and a puromycin medicine sieve is added after 48 hours. RNA was then extracted and qPCR tested to confirm good interference and overexpression efficiency (fig. 3c, d), and then cell-stable strains were used for subsequent functional experiments. Similarly, stable knockdown of LINC01419 by shRNA in Huh7 and PLC cells also significantly inhibited CCK8 proliferation, clonogenic, migratory and invasive capacity of hepatoma cells (fig. 5A-D). In contrast, overexpression of LINC01419 (Vector is an expression empty Vector control) in LINC 01419-low-expressed SK-Hep1 and HepG2 cells significantly promoted proliferation, clonogenic, migratory and invasive of hepatoma cells (fig. 5E-H). The above results all indicate the carcinomatous effect of LINC01419 in hepatoma cells.
2.4 in vivo experiments to investigate the relevance of LINC01419 to liver cancer cell proliferation, migration and invasion
Further carrying out in vivo animal experiments on the constructed cell strain of stable interference LINC01419, and carrying out 3×10 experiments 6 Subcutaneous injection of individual cells into 5-week-old female BALB/c nude mice a nude mice subcutaneous neoplasia model was constructed, and both independent LINC01419 knockdown groups of subcutaneous neoplasias grew at significantly lower rates and tumor weights than control groups (fig. 6a, b). Ki67 staining of tumor tissues also showed a significant decrease in Ki67 positive rate in the LINC01419 knockdown group of tumor cells (FIG. 6C).
At the same time construct stable expression EGFHuh7 cells of P-Luciferase and interfering LINC01419 on the basis of this, 1.5X10 were subjected to in vivo transfer experiments 6 The livers of individual cells injected into 5-week-old female BALB/c nude mice were used to construct a nude mice liver in situ transplantation tumor model, and after one month, live imaging was performed and the mice were euthanized and the liver and lung tissues of the mice were dissected. Imaging results showed that interference LINC01419 significantly inhibited liver cancer cell growth in situ in nude mice liver (fig. 6D), and HE staining and analysis of liver and lung tissue also showed that interference LINC01419 significantly inhibited tumor intrahepatic metastasis and decreased lung metastasis (fig. 6E). The above in vivo experiments further demonstrate the ability of LINC01419 to promote liver cancer cell growth and metastasis in vivo.
2.5 therapeutic Effect of GalNAc-silnc 01419 in vivo
Based on the specific high expression of LINC01419 in liver cancer and the normal liver not expression and the cancer promotion function exerted by LINC01419, the in vivo therapeutic effect and significance of GalNAc-siLINC01419 are further explored. GalNAc-siNC 01419 was subcutaneously injected into nude mice to evaluate the therapeutic effect of GalNAc-silnc 01419 in vivo by establishing a model for in situ xenograft of nude mice liver (see methods specifically, fig. 7). The results showed that the in vivo growth capacity of tumors was significantly reduced compared to control mice administered GalNAc-silnc 01419 treatment (fig. 8A-B); meanwhile, the degree of staining of Ki67 in tumor tissues of the GalNAc-silnc 01419 treated group mice was significantly reduced (FIG. 8C); in addition, by HE staining, galNAc-silnc 01419 treated mice were found to have significantly reduced intrahepatic and pulmonary transfer capacity compared to the control group (fig. 8D-E). The results indicate that the LINC01419 with high specificity and high expression of liver cancer cells can effectively inhibit the in-vivo growth and transfer capacity of nude mice xenograft tumors through GalNAc-siRNA targeting.
While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and the description is provided for clarity only, and those skilled in the art will recognize that the embodiments of the disclosure may be combined appropriately to form other embodiments that will be understood by those skilled in the art.
Claims (10)
1. Application of LINC 01419-targeted siRNA in preparing liver cancer drugs.
2. The use according to claim 1, wherein the targeting sequence of the siRNA comprises a nucleic acid set forth in SEQ ID No. 6.
3. The use according to claim 1 or 2, wherein the synthetic sequence of the siRNA comprises a nucleic acid shown in SEQ ID No. 15; preferably, the siRNA comprises a 2' -OMe modification (i 2 OMe) and a fluoro modification (i 2F); more preferably, the modified sequence is characterized by: i2OMeC// i2OMeC// i2OMeU// i2OMeA// i2OMeA// i2FU// i2OMeU// i2FU// i2FC// i2FC// i2FC// i2 OMeA' i2OMeU// i2OMeG// i2OMeG// i2OMeC// i2OMeA// i2OMeA// i2OMeU// i2OMeA// i2OMeU// i2OMeU// i2OMeU/.
4. The use according to any one of claims 1 to 3, wherein the 3' end of the siRNA is coupled to trivalent GalNAc.
5. The LINC 01419-targeted siRNA is characterized in that the targeting sequence of the siRNA comprises a nucleic acid shown in SEQ ID NO. 6.
6. The siRNA of claim 5, wherein the siRNA comprises a 2' -OMe modification (i 2 OMe) and a fluoro modification (i 2F); preferably, the specific modified sequences are characterized by: i2OMeC// i2OMeC// i2OMeU// i2OMeA// i2OMeA// i2FU// i2OMeU// i2FU// i2FC// i2FC// i2FC// i2 OMeA' i2OMeU// i2OMeG// i2OMeG// i2OMeC// i2OMeA// i2OMeA// i2OMeU// i2OMeA// i2OMeU// i2OMeU// i2OMeU/.
7. siRNA according to claim 5 or 6, characterized in that the 3' end of the siRNA is coupled to trivalent GalNAc.
8. Application of a reagent for detecting LINC01419 level in preparing liver cancer prognosis evaluation kit.
9. The use according to claim 8, wherein the reagent comprises primers and/or probes for LINC01419 detection.
10. The use according to claim 9, wherein the primers for LINC01419 are shown in SEQ ID NO. 3 and 4.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488722A (en) * | 2017-09-18 | 2017-12-19 | 复旦大学附属中山医院 | Purposes of the LncRNA in the reagent for preparing diagnosis adenocarcinoma of lung |
| CN108588220A (en) * | 2018-04-26 | 2018-09-28 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma long-chain non-coding RNA LINC01419 molecular markers and its application |
| CN111485020A (en) * | 2020-04-15 | 2020-08-04 | 湖南省科域生物医药科技有限公司 | L INC01419 application as marker for diagnosing and treating liver cell cancer |
| CN113286895A (en) * | 2018-09-05 | 2021-08-20 | 阿莫内塔诊断股份公司 | Long non-coding RNA (lncRNA) for the diagnosis and treatment of brain disorders, in particular cognitive disorders |
-
2023
- 2023-10-19 CN CN202311362809.4A patent/CN117653653A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488722A (en) * | 2017-09-18 | 2017-12-19 | 复旦大学附属中山医院 | Purposes of the LncRNA in the reagent for preparing diagnosis adenocarcinoma of lung |
| CN108588220A (en) * | 2018-04-26 | 2018-09-28 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma long-chain non-coding RNA LINC01419 molecular markers and its application |
| CN113286895A (en) * | 2018-09-05 | 2021-08-20 | 阿莫内塔诊断股份公司 | Long non-coding RNA (lncRNA) for the diagnosis and treatment of brain disorders, in particular cognitive disorders |
| CN111485020A (en) * | 2020-04-15 | 2020-08-04 | 湖南省科域生物医药科技有限公司 | L INC01419 application as marker for diagnosing and treating liver cell cancer |
Non-Patent Citations (8)
| Title |
|---|
| GONG ZHANG等: "LINC01419 facilitates hepatocellular carcinoma growth and metastasis through targeting EZH2-regulated RECK", AGING, vol. 12, no. 11, 10 June 2020 (2020-06-10), pages 11071 - 11081 * |
| ZENG-PENG SUN 等: "Long noncoding RNA LINC01419 promotes hepatocellular carcinoma malignancy by mediating miR-485-5p/LSM4 axis", J MED SCI., 31 December 2022 (2022-12-31), pages 826 - 838 * |
| ZIMING CHENG 等: "LINC01419 promotes cell proliferation and metastasis in lung adenocarcinoma via sponging miR-519b-3p to up-regulate RCCD1", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 30 September 2019 (2019-09-30), pages 107 - 114, XP085867975, DOI: 10.1016/j.bbrc.2019.09.090 * |
| 丁洁 等: "微RNA 在肝癌中的作用及其应用前景", 生命的化学, vol. 29, no. 2, 31 December 2009 (2009-12-31), pages 243 - 246 * |
| 丁瑞: "长链非编码RNA LINC01419与肝细胞癌增殖和侵袭的相关性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, 14 January 2022 (2022-01-14), pages 8 - 11 * |
| 方伟兰: "LINC01419通过调控miR-503-5p促进肝细胞癌的增殖、迁移和侵袭", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, 15 February 2023 (2023-02-15), pages 17 - 21 * |
| 程秀芝: "长链非编码RNA LINC01419在肝细胞癌中作用及机制的初步研究", 中国优秀硕士学位论文全文数据库 医药卫生科技辑, 15 January 2022 (2022-01-15), pages 072 - 1239 * |
| 苟苗苗 等: "LncRNAs在肝细胞癌中的研究进展", 解放军医学院学报, vol. 40, no. 03, 19 December 2018 (2018-12-19), pages 293 - 297 * |
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